Project A: Establish the role of factors regulating PPi/Pi levels, e.g., ANK, NPP1, PHOSPHO1, and TNAP, in root formation and cementogenesis and apply this knowledge to deliver factors locally/systemically to regenerate periodontal tissues, using rodent models of periodontal disease. Results demonstrate the importance of regulators of Pi/PPi and of functional SIBLING family genes/proteins during mineralization, highlighting the need for fine tuning physiochemical/cellular molecular factors toward achieving a homeostatic balance required for formation/regeneration of periodontal tissues. 1. Mutations in ALPL result in hypophosphatasia (HPP), a disease causing defective skeletal mineralization. ALPL encodes tissue nonspecific alkaline phosphatase (TNAP), an enzyme that promotes mineralization by reducing pyrophosphate (PPi), a mineralization inhibitor. A novel mouse model for tooth-specific HPP (odonto-HPP) was created by knocking in an autosomal dominant ALPL missense mutation (causing a base change, A116T), which was identified in a human kindred. Alpl+/A116T mice featured alterations in the alveolar bone, including radiolucencies and resorptive lesions, osteoid accumulation on the alveolar bone crest, and significant differences in several bone properties measured by microCT. Non-significant changes in acellular cementum did not appear to affect periodontal attachment or function, though systemic ALP correlated significantly with incisor cementum thickness. The Alpl+/A116T mouse is the first model of odontohypophosphatasia, providing insights on dentoalveolar development and function and providing a new model for testing potential dental-targeted therapies in future studies. Foster et al 2016 2. Phospho1 KO and AlplxPhospho1 dKO: Phospho1 KO mice featured modest disturbances in mineralization of alveolar bone, mantle dentin, and cellular cementum, yet acellular cementum and PDL appeared undisturbed. In contrast to the skeleton, genetic ablation of Spp1 (gene encoding OPN) did not ameliorate qualitative or quantitative defects in Phospho1 KO dentoalveolar tissues. These data support a role for PHOSPHO1 in proper mineralization of alveolar bone, dentin, and cellular cementum and that matrix vesicles may not be required for acellular cementum formation. Zweifler et al. 2016 3. ANK, NPP1 and TNAP in vivo and vitro: Examining Ank and Npp1 KO animals revealed a compensatory mechanism related to gene expression. Also, the expression patterns and functions of TNAP and NPP1 in cementum mineralization suggest that the early expression of TNAP creates a low PPi environment allowing for AEFC initiation, while later expression of NPP1 increases Ppi, restricting AEFC apposition. These results provide the rationale for our in vivo studies (to begin late 2016) using periodontal wound healing models in rodents to deliver factors controlling Pi/Ppi, using lentivirus constructs. 4. In an effort to define specific functions for ANK vs ENPP1, we are developing a double KO (dKO). We will reevaluate outcomes and next steps in Oct. 2016. Project B: Defining the role of extracellular matrix proteins in periodontogenesis with a focus on SIBLINGS and collagen. BSP: BSP belongs to the Small Integrin Binding Ligand N-linked Glycoprotein (SIBLING) family, a family of acidic phosphoproteins, expressed in mineralized tissues. We reported that BSP KO mice have a profound periodontal phenotype, similar to Alpl KO mice. Building on this, in collaboration with Dr. H. Goldberg, we examined tendon/ligament function in KO mice and reported that, based on the known in vitro functional properties of the protein, BSP may be a useful therapeutic molecule in the reattachment of tendons and ligaments to bone. Marinovich et al. 2016 OPN: We identified OPN, a SIBLING, as a factor of interest based on proteomic analysis of PDL from Ank-/- mice. Several models were used to determine OPNs function with the PDL region. We conclude that OPN does not have a non-redundant or critical role in regulating cementum mineralization and periodontal structure and function during development. Yet, in wound healing models loss of OPN function may have consequences, which is being explored by several groups at this time. Plan to submit manuscript Sept.2016 Ongoing: To further define the mechanistic aspects of BSPs regulation of genes associated with Pi/PPi modulation, Bsp KO cementoblasts (OCCM30), using CRISPR CAS technology, were generated. Preliminary results suggest that BSP may have at least two roles in the mineralization process, i.e., regulating modulators of local Pi/PPi levels and regulating expression of transcription factors through various feedback loops. In addition, we are developing an OPN x BSP dKO to determine if OPN KO would alter the BSP KO phenotype. Collagen: Logic would suggest that individuals with osteogenesis imperfect (OI), a heritable disorder of the extracellular matrix characterized by low bone mass, brittle and fragile bones, and bone fractures, would have marked periodontal defects. Yet, reports to date focus on general bone pathology and, in some cases, dentin defects (dentinogenesis imperfecta (DI)), with limited attention to the periodontium. Undertaking a multi-collaborative project, with several extramural investigators as part of the Brittle Bone Consortium, has enabled us to obtain tissues from four types of OI mice as well as extracted teeth from OI patients. For the first time, we identify periodontal changes in mice resulting from autosomal recessive and autosomal dominant OI. Changes reflect developmental defects, as well as altered function and remodeling during later stages in life. Ongoing: Using CRISPR/Cas9 technologies and OCCM cells we will prepare Knock-in c.1546G &RT; T cells (mimic Brtl+/- mouse) and Crtap complete gene KO to define the effects of these modifications on cell function. Other projects: IRF6, CL/P and dental manifestations: Emily Chu DDS, PhD for fulfillment of her PhD. (Will not be continued at NIAMS/NIH). Clefting of the lip, with or without palatal involvement (CLP), is associated with a higher incidence of developmental tooth abnormalities. Because most CLP genes are expressed throughout the oral epithelium, we hypothesized that CLP genes play an important functional role in tooth patterning and amelogenesis. Results support a role for IRF6 in tooth number, crown and root morphology and amelogenesis that is likely due to a functional role of Irf6 in organization and polarity of epithelial cell types. Our data reinforce the notion that various isolated tooth defects could be considered part of the CLP spectrum in relatives of an affected individual. Chu et al. 2016 In addition, we have established a new immortalized cell line: Murine Cementocytes in collaboration with Lynda Bonewald. Zhao et al. 2016